Transfection reagents

ABSTRACT

Disclosed are compounds capable of facilitating transport of biologically active agents or substances into cells having the general structure: 
     
       
         
         
             
             
         
       
     
     wherein Q is selected from the group consisting of N, O and S; L is any bivalent organic radical capable of linking each Q, such as C, CH, (CH 2 ) l , or {(CH 2 ) i —Y—(CH 2 ) j } k , wherein Y is selected from the group consisting of CH 2 , an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, a carbonyl, and a secondary amino group and wherein Y is optionally substituted by —X 1 -L′-X 2 —Z or —Z; R 1  R 6 , independently of one another, are selected from the group consisting of H, —(CH 2 ) p -D-Z, an alkyl, an alkenyl, an aryl, and an alkyl or alkyl ether optionally substituted by one or more of an alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, a guanidyl, or a carbamoyl group, and wherein at least one of R 1 , R 3 , R 4  and R 6  is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group; and anyone of R 1 , R 3 , R 4  and/or R 6  may optionally be covalently linked with each other, with Y or with L when L is C or CH to form a cyclic moiety; Z is selected from the group consisting of amine, spermiyl, carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, and protein; X 1  and X 2 , independently of one another, are selected from the group consisting of NH, O, S, alkylene, and arylene; L′ is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, alkylene ether, and polyether; D is Q or a bond; A 1  and A 2 , independently of one another, are selected from the group consisting of CH 2 O, CH 2 S, CH 2 NH, C(O), C(NH), C(S) and (CH 2 )t; X is a physiologically acceptable anion; m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both m and n are 0 at least one of r, s, u and y is other than 0; i, j, k, l, p and are integers from 0 to about 100; q is an integer from 1 to about 1000; and a is the number of positive charge divided by the valence of the anion.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/420,091, filed Mar. 14, 2012, which application is a continuation ofU.S. application Ser. No. 12/353,371 filed Jan. 14, 2009, whichapplication is a continuation of U.S. application Ser. No. 11/040,687,filed Jan. 21, 2005, now U.S. Pat. No. 7,479,573, which is acontinuation of U.S. application Ser. No. 09/438,365, filed Nov. 12,1999, now U.S. Pat. No. 7,166,745, which claims the benefit under 35U.S.C. §119(e) of U.S. provisional application Ser. No. 60/108,117,filed Nov. 12, 1998, the disclosures of which applications areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to cationic lipids and compositions ofcationic lipids having utility in lipid aggregates for delivery ofmacromolecules and other compounds into cells.

2. Related Art

Lipid aggregates such as liposomes have been found to be useful asagents for delivery to introduce macromolecules, such as DNA, RNA,protein, and small chemical compounds such as pharmaceuticals, to cells.In particular, lipid aggregates comprising cationic lipid componentshave been shown to be especially effective for delivering anionicmolecules to cells. In part, the effectiveness of cationic lipids isthought to result from enhanced affinity for cells, many of which bear anet negative charge. Also in part, the net positive charge on lipidaggregates comprising a cationic lipid enables the aggregate to bindpolyanions, such as nucleic acids. Lipid aggregates containing DNA areknown to be effective agents for efficient transfection of target cells.

The structure of various types of lipid aggregates varies, depending oncomposition and method of forming the aggregate. Such aggregates includeliposomes, unilamellar vesicles, multilameller vesicles, micelles andthe like, having particular sizes in the nanometer to micrometer range.Methods of making lipid aggregates are by now well-known in the art. Themain drawback to use of conventional phospholipid containing liposomesfor delivery is that the material to be delivered must be encapsulatedand the liposome composition has a net negative charge which is notattracted to the negatively charged cell surface. By combining cationiclipid compounds with a phospholipid, positively charged vesicles andother types of lipid aggregates can bind DNA, which is negativelycharged, can be taken up by target cells, and can transfect targetcells. (Felgner, P. L. et al. (1987) Proc. Natl. Acad. Sci. USA84:7413-7417, Eppstein, D. et al., U.S. Pat. No. 4,897,355.)

A well-known cationic lipid isN-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).The structure of DOTMA is:

DOTMA by itself or in 1:1 combination withdioleoylphosphatidylethanolamine (DOPE) is formulated into liposomesusing standard techniques. Feigner, et al. supra demonstrated that suchliposomes provided efficient delivery of nucleic acids to some types ofcells. A DOTMA:DOPE (1:1) formulation is sold under the trade nameLIPOFECTIN (Life Technologies. Inc., Rockville, Md.). Anothercommercially available cationic lipid is1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), which differsfrom DOTMA only in that the oleoyl moieties are linked via ester, ratherthan ether bonds to the propylamine. A related group of compounds differfrom DOTMA and DOTAP in that one of the methyl groups of thetrimethylammonium group is replaced by a hydroxyethyl group. Compoundsof this type are similar to the Rosenthal Inhibitor (RI) ofphospholipase A (Rosenthal, A. F. and Geyer, R. P. (1960) J. Biol. Chem.235:2202-2206) which has stearoyl esters linked to the propylamine core.The dioleoyl analogs of RI are commonly abbreviated as DORI-ether andDORI-ester, depending on the linkage of the fatty acid moieties to thepropylamine core. The hydroxy group can be used as a site for furtherfunctionalization.

The dimyristyloxy analog of RI is known as DRMIE. A 1:1 (M/M)DMRIE:cholesterol formulation is sold under the trade name DMRIE-C (LifeTechnologies, Inc., Rockville. Md.). The structure of DMRIE is:

Another class of compounds has been disclosed by Behr et al. (1989)Proc. Natl. Acad. Sci. USA 86:6982-6986; EPO publication 0 394 111 (Oct.24, 1990), in which carboxyspermine has been conjugated to two types oflipids. The structure of 5-carboxyspermylglycine dioctadecylamide (DOGS)is:

The structure of dipalmitoylphosphatidylethanolamine5-carboxyspermylamide (DPPES) is:

Both DOGS and DPPES have been used to coat plasmids, forming a lipidaggregate complex that provides efficient transfection. The compoundsare claimed to be more efficient and less toxic than DOTMA fortransfection of some cell lines. DOGS is available commercially asTRANSFECTAM™ (Promega. Madison. Wis.).

Another class of compounds has been also described in which carboxyspermine has been conjugated to lipids via an amide bond (Gebeyehu, G.et al., U.S. Pat. No. 5,334,761). These compounds are useful for anefficient delivery of nucleic acids into various cells and also areintermediates for making other such lipids.2,3-di-oleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propan-aminium(DOSP A) is available as a 3:1 (w/w) formulation with DOPE under thetrade name LIPOFECTAMINE® (available from Life Technologies, Inc.,Rockville, Md.). The structure of DOSPA is as follows:

Lipid compounds with a spermine head group have also been described(Haces, A., et al., U.S. Pat. No. 5,674,908). These compounds areespecially useful for delivery of nucleic acids into insect cells. A1:1.5 (M/M) formulation of tetramethyltetrapalmitylspermine (TM-TPS) toDOPE is commercially available under the trade name CELLFECTIN® (LifeTechnologies, Inc., Rockville, Md.). The structure of TM-TPS is shownbelow:

A cationic cholesterol derivative (DC-Chol) has been synthesized andformulated into liposomes in combination with DOPE. (Gao, X. and Huang,L. (1991) Biochim. Res. Comm. 179:280-285). The compound's structure is:

Liposomes formulated with DC-Chol are said to provide more efficienttransfection and lower toxicity than DOTMA-containing liposomes for somecell lines.

Lipopolylysine, formed by conjugating polytysine to DOPE, has beenreported to be especially effective for transfection in the presence ofserum, a condition likely to be encountered in vivo (Zhou, X. et al.(1991) Biochim. Biophys. Acta 1065: 8-14).

Despite advances in the field, a need remains for a variety of improvedcationic lipid compounds. In particular, no single cationic lipid todate has been found to work well with all cell types. Since differentcell types differ from one another in membrane composition, it is notsurprising that different compositions and types of lipid aggregates areeffective for different cell types, either for their ability to contactand fuse with target cell membranes, or for aspects of the transferprocess itself. At present these processes are not well understood,consequently the design of effective liposomal precursors is largelyempirical. Besides content and transfer, other factors are ofimportance, for example, ability to form lipid aggregates suited to theintended purpose, the possibility of transfecting cells in the presenceof serum-toxicity to the target cell, stability as a carrier for thecompound to be delivered, and ability to function in an in vivoenvironment. In addition, lipid aggregates can be improved by broadeningthe range of substances which can be delivered to cells. The cationiclipid compounds of the present invention have improved function withrespect to several of the foregoing attributes.

SUMMARY OF THE INVENTION

The present invention provides novel cationic lipids according to thegeneral Formula (A):

wherein

Q is selected from the group consisting of N, O and S; L is any bivalentorganic radical capable of covatently linking each Q, such as C, CH,(CH₂)_(l) or {(CH₂)_(i)—Y-{(CH₂)_(j)}_(k), wherein Y is selected fromthe group consisting of CH₂, an ether, a polyether, an amide, apolyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, acarbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, acarbonyl, and a secondary amino group and wherein Y is optionallysubstituted by —X₁-L′-X₂—Z or —Z;

R₁-R₆, independently of one another, are selected from the groupconsisting of H, —{CH₂)_(p)-D-Z, an alkyl, an alkenyl, an aryl, and analkyl or alkyl ether optionally substituted by one or more of analcohol, an amino alcohol, an amine, an amide, an ether, a polyether, apolyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, aguanidyl, or a carbamoyl group, and wherein at least one of R₁, R₃, R₄and R₅ is a straight chain or branched, cyclic, alkyl, alkenyl, alkynylor aryl group; and any one or more of R₁, R₃, R₄ and R₆ may optionallybe covalently linked with each other, with Y or with L when L is C or CHto form a cyclic moiety;

Z is selected from the group consisting of amine, spermiyl,carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl,pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, andprotein;

X₁ and X₂, independently of one another, are selected from the groupconsisting of NH, O, S, alkylene, and arylene;

L′ is selected from the group consisting of alkylene, alkenylene,alkynylene, arylene, alkylene ether, and polyether;

D is Q or a bond;

A₁ and A₂, independently of one another, are selected from the groupconsisting of CH₂O, CH₂S, CH₂NH, C(O), C(NH), C(S) and (CH₂)_(t);

X is a physiologically acceptable anion:

m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both mand n are 0 at least one of r, s, u and y is other than 0;

i, j, k, l, p and t are from a to about 100;

q is an integer from 1 to about 1000; and

a is the number of positive charge divided by the valence of the anion.

Further, the present invention provides novel cationic lipids accordingto the general Formula (B):

wherein

L is (CH₂)_(l) or {(CH₂)_(i)—Y—(CH₂)_(j)}_(k) wherein Y is selected fromthe group consisting of an ether, a polyether, an amide, a polyamide, anester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, acarbonate, and a secondary amino group;

R₁-R₆, independently of one another, are selected from the groupconsisting of H, —(CH_(2p)—Z, an alkyl, an alkenyl, an aryl, and analkyl or an alkyl ether optionally substituted by one or more of analcohol, an aminoalcohol an amine, an amide, an ether, a polyether, apolyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or acarbamoyl group, and at least one of R₁, R₃, R₄ and R₆ is a straightchain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group,preferably having from about 2 to 100, preferably 4 to 75, morepreferably 6 to 64, more preferably 8 to 50, more preferably 8 to 40,more preferably 8 to 30, more preferably 6 to 30, more preferably 4 to30, more preferably 2 to 30, and most preferably 8 to about 24 carbonatoms, and anyone or more of R₁, R₃, R₄ and/or R₆ may optionally becovalently linked with each other to form a cyclic moiety;

Z is selected from the group consisting of amine, spermiyl,carboxyspermiyl guanidyl, spemlidinyl, putricinyl, diaminoalkyl,pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, amino acidderivative, peptide, and protein;

A₁ and A₂, independently of one another, are selected from the groupconsisting of CH₂O, CH₂S, CH₂NH, C(O), C(NH), C(S) and (CH₂)t;

X is a physiologically acceptable anion, such as the halide anions,chloride, bromide, and iodide as well as acetate, sulfate,trifluoroacetate, etc.;

m, n, v and w are 0 or 1;

i, j, k, l, p and t are integers from 1 to about 100, more preferably 1to 50, more preferably 1 to 25, more preferably 1 to 15, more preferably1 to 10 and most preferably 1 to about 4;

q is an integer from 1 to about 1000, preferably from 1 to about 500,more preferably from 1 to about 250, more preferably from 1 to about100, more preferably from 1 to about 50, more preferably from 1 to about25, more preferably from 1 to about 12, most preferably from 1 to about6; and

a is the number of positive charges divided by the valence of the anion,wherein when m and n are 0, then a is O.

Also, the present invention provides novel cationic lipids according tothe Formula (C):

wherein

Y is selected from the group consisting of CH₂, an ether, a polyether,an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, aguanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide,an imine, a carbonyl, and a secondary amino group and wherein Y isoptionally substituted by —X₁-L′-X₂—Z or —Z;

R₁, R₃, R₄ and R₆, independently of one another, are selected from thegroup consisting of H, —{CH₂)_(p)-D-Z, an alkyl, an alkenyl, an aryl,and an alkyl or an alkyl ether optionally substituted by one or more ofan alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether,a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, aguanidyl, or a carbamoyl group, and at least one of R₁, R₃, R₄ and R₆ isa straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or arylgroup, most preferably having from about 8 to about 24 carbon atoms, andR₁, R₃, R₄ and R₅ may optionally be covalently linked with each other orwith Y, to form a cyclic moiety;

Z is selected from the group consisting of amine, spemliyl,carboxyspemliyl, guanidyl, spemlidinyl, putricinyl, diaminoalkyl,pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, andprotein:

X₁ and X₂, independently of one another, are selected from the groupconsisting of NH, O, S, alkylene, and arylene;

L′ is selected from the group consisting of alkylene, alkenylene,alkynylene, arylene, alkylene ether, and polyether;

D is Q or a bond;

m and n are 0 or 1; and

i, j, k, l and p are integers from 1 to about 10.

Further, the present invention provides compounds or polycationsaccording to the Formula (D):

wherein

L is C, CH, (CH₂)_(l) or {(CH₂)_(i)—Y—(CH₂)_(j)}_(k), wherein Y isselected from the group consisting of CH₂, an ether, a polyether, anamide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl,a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine,a carbonyl, and a secondary amino group and wherein Y is optionallysubstituted by —X₁-L′-X₂—Z or —Z:

R₁-R₆, independently of one another, are selected from the groupconsisting of H, —(CH₂)_(p)-D-Z, an alkyl, an alkenyl, an aryl, and analkyl or an alkyl ether optionally substituted by one or more of analcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, apolyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, aguanidyl, or a carbamoyl group, and wherein at least one of R₁, R₃, R₄and R₆ is a straight chain or branched, cyclic, alkyl, alkenyl, alkynylor aryl groups, preferably having from about 2 to about 30 carbon atoms,more preferably from 8 to 24 carbon atoms;

Z is selected from the group consisting of amine, spenniyl,carboxyspenniyl, guanidyl, spennidinyl, putricinyl, diaminoalkyl,pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, amino acidderivative, peptide, and protein;

X₁ and X₂, independently of one another, are selected from the groupconsisting of NH, O, S, alkylene and arylene;

L′ is selected from the group consisting of alkylene, alkenylene,alkynylene, arylene, alkylene ether, and polyether;

A₁ and A₂, independently of one another, are selected from the groupconsisting of CH₂O, CH₂S, CH₂NH, C(O), C{NH), C(S) and (CH₂)t;

m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both mand n are 0 at least one of r, s, u and y is other than 0;

i, j, k, l, p and t are integers from 0 to about 100; and

q is an integer from 1 to about 1000.

Also, the present invention provides compounds or polycations accordingto the Formula (E):

wherein

L is (CH₂)_(l) or {(CH₂)_(i)—Y—(CH₂)_(j)}_(k), wherein Y is selectedfrom the group consisting of CH₂, an ether, a polyether, an amide, apolyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, acarbamoyl, a carbonate, and a secondary amino group;

R₁, R₆, independently of one another, are selected from the groupconsisting of H, —(CH₂)_(p)—Z, an alkyl, an alkenyl, an aryl, and analkyl or an alkyl ether optionally substituted by one or more of analcohol, an amino alcohol, an amine, an amide, an ether, a polyether, apolyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or acarbamoyl group, and at least one of R₁, R₃, R₄ and R₆ is a straightchain or branched, cyclic, alkyl, alkenyl, alkenyl or aryl group,preferably having from about 2 to about 30 carbon atoms, more preferablyhaving from about 8 to about 24 carbon atoms;

Z is selected from the group consisting of amine, spenniyl,carboxyspenniyl, guanidyl, spennidinyl, putricinyl, diaminoalkyl,pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, amino acidderivative, peptide, and protein;

A₁ and A₂, independently of one another, are selected from the groupconsisting of CH₂O, CH₂S, CH₂NH, C(O), C(NH), C(S) and (CH₂)t;

m, n, v and w are 0 or 1;

i, j, k, l, p and t are integers from 1 to about 100; and

q is an integer from 1 to about 1000.

Also, the present invention provides novel compounds falling within thescope of the above formulae.

The compounds of the invention are useful, either alone or incombination with other lipid aggregate-forming components (e.g., DOPE,DOPC or cholesterol) for formulation into liposomes or other lipidaggregates. Such aggregates are polycationic, able to form stablecomplexes with anionic macromolecules, such as nucleic acids. The lipidaggregate macromolecular complex interacts with cells making thepolyanionic macromolecule available for absorption and uptake by thecell.

The present invention provides a lipid aggregate comprising one or moreof the compounds of the present invention. Preferably, the lipidaggregate comprises at least one lipid aggregate-forming compound.Preferably, the lipid aggregate-forming compound is selected from thegroup consisting of DOPE, DOPC and cholesterol.

The compounds of the present invention may also be conjugated to ormixed with or used in conjunction with a variety of useful molecules andsubstances such as proteins, peptides, growth factors and the like toenhance cell-targeting, uptake, internalization, nuclear targeting andexpression.

This invention also includes lipid aggregates comprising one or morecompounds of the present invention or mixtures thereof. Such lipidaggregates may be combined with one or more aggregate-forming componentsand/or transfection enhancers.

The transfection methods of the present invention employing thecompounds or compositions (such as those described above) of the presentinvention or mixtures thereof can be applied to in vitro and in vivotransfection of cells, particularly to transfection of eukaryotic cellsor tissues including animal cells, human cells, insect cells, plantcells, avian cells, fish cells, mammalian cells and the like.

Accordingly, the present invention provides a method for introducing apolyanion into a cell or cells, wherein the method comprises forming aliposome from a positively charged compound according to the invention,contacting the liposome with polyanion to form a positively-chargedpolyanion-liposome complex and incubating the complex with a cell orcells.

The methods of this invention can be used to generate transfected cellsor tissues which express useful gene products. The methods of thisinvention can also be used as a step in the production of transgenicanimals. The methods of this invention are useful in any therapeuticmethod requiring introducing of nucleic acids into cells or tissues. Inparticular, these methods are useful in cancer treatment, in in vivo andex vivo gene therapy, and in diagnostic methods. See, for example, U.S.Pat. No. 5,589,466 to Felgner, et al. and U.S. patent application Ser.No. 08/450,555 filed on May 25, 1995 to Jessee, et al. The transfectioncompounds or compositions of this invention can be employed as researchreagents in any transfection of cells or tissues done for researchpurposes. Nucleic acids that can be transfected by the methods of thisinvention include DNA and RNA from any source comprising natural basesor non-natural bases, and include those encoding and capable ofexpressing therapeutic or otherwise useful proteins in cells or tissues,those which inhibit expression of nucleic acids in cells or tissues,those which inhibit enzymatic activity or activate enzymes, those whichcatalyze reactions (ribozymes), and those which function in diagnosticassays.

The compounds, compositions and methods provided herein can also bereadily adapted in view of the disclosure herein to introducebiologically active macromolecules or substances other than nucleicacids, including, among others, polyamines, polyamine acids,polypeptides, proteins, biotin, and polysaccharides into cells. Otheruseful materials for example, therapeutic agents, diagnostic materialsand research reagents, can be introduced into cells by the methods ofthis invention. In a preferred aspect, any nucleic acid vector may bedelivered to or into a cell by the present invention.

Accordingly, the present invention provides a method for introducing abiologically active substance into a cell, wherein the method comprisesforming a liposome of a compound according to the invention and abiologically active substance and incubating the liposome with a cell orcell culture.

The invention also relates to compositions comprising the compounds ofthe invention and one or more additional components selected from thegroup consisting of nucleic acids, cells, buffers, culture media,biologically active substance, neutral lipids, and transfectionenhancers, preferably a nucleic acid.

This invention also includes transfection kits which include one or moreof the compounds or compositions of the present invention or mixturesthereof. Particularly, the invention provides a kit comprising one ormore of the compounds of the present invention and at least oneadditional component selected from the group consisting of a cell,cells, a cell culture media, a nucleic acid, a transfection enhancer andinstructions for transfecting a cell or cells.

The invention also relates to intermediates and methods for using suchintermediates for making the compounds or compositions of the invention.The invention also relates to the compositions, compounds or componentsobtained by the interaction of materials (intermediates, compounds,lipids etc.) used in the synthesis methods of the invention.

Other preferred embodiments of the present invention will be apparent toone of ordinary skill in the art in view of the following drawings anddescription of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the transfection of HEK-293 cells withcationic transfection reagents.

FIG. 2 is a graph showing transfection of COS-7 cells with cationictransfection reagents.

FIG. 3 is a graph showing transfection of CHO-KI cells with cationictransfection reagents.

FIG. 4 is a graph showing transfection of He La cells with cationictransfection reagents.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to cationic lipids and compositions ofcationic lipids having utility in lipid aggregates for delivery ofmacromolecules and other compounds into cells. The compounds can be usedalone or in combination with other compounds to prepare liposomes andother lipid aggregates suitable for transfection or delivery ofcompounds to target cells, either in vitro or in vivo.

The compounds of the present invention are preferably polycationic andpreferably thus form highly stable complexes with various anionicmacromolecules, particularly polyanions such as nucleic acids. Thesecompounds have the property, when dispersed in water, of forming lipidaggregates which associate strongly, via their cationic portion, withpolyanions. By using an excess of cationic charges relative to theanionic compound, the polyanion-lipid complexes may be adsorbed on cellmembranes, thereby facilitating uptake of the desired compound by thecells.

The present invention also relates to intermediates for preparing thecompound and compositions of the invention.

More specifically, the present invention relates to a cationic lipid fortransfection which has a greater transfection efficiency thancommercially available products in the three most common cell types usedin expression research (CHO-KI, COS-7, and HEK293) making it useful forhigh throughput applications; and which has a simple to use protocol asdefined by the fact that no additional reagents are required (e.g., suchas 10 LIPOFECTAMINE® PLUS™ Reagent available from Life Technologies,Inc., Rockville, Md.), no removal of serum and therefore no mediachanges are required, and the DNA/lipid complex do not need to beremoved from the cells prior to assay.

The compounds according to the present invention have the Formula (A):

wherein

Q is selected from the group consisting of N, O and S;

L is any bivalent organic radical capable of covalently linking each Q,such as C, CH, (CH₂)_(l) or {(CH₂)_(i)—Y—(CH₂)_(j)}_(k), wherein Y isselected from the group consisting of CH₂, an ether, a polyether, anamide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl,a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine,a carbonyl, and a secondary amino group and wherein Y is optionallysubstituted by —X₁-L′-X₂—Z or —Z;

R₁ R₆, independently of one another, are selected from the groupconsisting of H, —(CH₂)_(p)-D-Z, an alkyl, an alkenyl, an aryl, and analkyl or alkyl ether optionally substituted by one or more of analcohol, an amino alcohol, an amine, an amide, an ether, a polyether, apolyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, aguanidyl, or a carbamoyl group, and wherein at least one of R₁, R₃, R₄and R₆ is a straight chain or branched, cyclic, alkyl, alkenyl, alkynylor aryl group; and R₁ and R₄ or R₃ and R₆ may optionally be covalentlylinked with each other, with Y or with L when L is C or CH to form acyclic moiety;

Z is selected from the group consisting of amine, spermiyl,carboxyspermiyl, guanidyl, spennidinyl, putricinyl, diaminoalkyl,pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, andprotein;

X₁ and X₂, independently of one another, are selected from the groupconsisting of NH, O, S, alkylene, and arylene;

L′ is selected from the group consisting of alkylene, alkenylene,alkynylene, arylene, alkylene ether, and polyether;

D is Q or a bond;

A₁ and A₂, independently of one another, are selected from the groupconsisting of CH₂O, CH₂S, CH₂NH, C(O), C(NH), C(S) and (CH₂)t;

X is a physiologically acceptable anion;

m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both mand n are 0 at least one of r, s, u and y is other than 0;

i, j, k, l, p and t are integers from 0 to about 100;

q is an integer from 1 to about 1000; and

a is the number of positive charge divided by the valence of the anion.

Preferably the alkyl ether optionally substituted by one or more alcoholgroups comprises a carbohydrate. Preferably, the carbohydrate isselected from the group consisting of galactose, fructose, glucose,maltose, sucrose, cellobiose, lactose, mannose, glucopyranose,mannopyranose and galactopyranose.

Preferably, i, j, k, l, p and t are integers independently selected from1 to 100, more preferably from 1 to 50, more preferably 1 to 25, morepreferably 1 to 15, more preferably 1 to 10 and most preferably 1 toabout 4. Preferably, 1, b and c are integers from 1 to about 4, i and jare integers from about 2 to about 3 and k is an integer from 1 to about3.

Preferably, q is an integer from 1 to about 500, more preferably from 1to about 250, more preferably from 1 to about 100, more preferably from1 to about 50, more preferably from 1 to about 25, more preferably from1 to about 12, most preferably from 1 to about 6.

Preferably, at least one of R₁, R₃, R₄ and R₆ is a straight chain orbranched, cyclic, alkyl, alkenyl, alkynyl or aryl group having fromabout 2 to 100, preferably 4 to 75, more preferably 6 to 64, morepreferably 8 to 50, more preferably 8 to 40, more preferably 8 to 30,more preferably 6 to 30, more preferably 4 to 30, and most preferably 8to about 24 carbon atoms.

In all aspects of the invention, most suitable R1 and R4 groups, whichcan be the same or different, preferably the same, are C₆₋₃₀ hydrocarbonradicals derived from fatty acids or activated derivatives thereof, suchas fatty acyl chlorides. Thus, typical R₁ and R₄ groups are C₆₋₃₀ alkylor alkenyl groups.

Preferably, R₁, R₂, R₃, R₄, R₅ and R₆, independently of one another, areselected from the group consisting of H, C₁-C₈ alkyl, alkenyl, aryl, andalkyl optionally substituted by one or more of an alcohol, an amide, anether, a polyether, a polyamide, an ester, a mercaptan, a urea, athiourea, a guanidyl, or a carbamoyl group, and at least one of R₁, R₃,R₄ and R₆ is a straight chain or branched, cyclic, alkyl, alkenyl,alkynyl or aryl group having from about 8 to about 24 carbon atoms.

Preferably Q is N.

Preferably, Y is selected from the group consisting of CH₂, O, S and NH.

Useful compounds falling within the scope of the above formula (A)include compounds having the following formulae:

wherein

Q and L are as defined above;

R₁, R₃, R₄ and R₆, independently of one another, are selected from thegroup consisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyloptionally substituted by one or more of an alcohol, an amine, an amide,an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, athiourea, a guanidyl, or a carbamoyl group;

r, s, u and y are 0 or 1; and

R₇ and R₈ are independently H or a carbohydrate;

wherein

Q is as defined above;

R₁, R₂, R₄ and R₅, independently of one another, are selected from thegroup consisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyloptionally substituted by one or more of an alcohol, an amine, an amide,an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, athiourea, a guanidyl, or a carbamoyl group;

Z is selected from the group consisting of spermiyl, spermidiyl, aminoacid, peptidyl, diaminoalkyl, and polyamine;

m, n, r and u are 0 or 1; and

l, b and c are integers independently selected from 1 to about 4;

wherein

Q, R₁, R₄, m, n, r and u are as defined above;

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

Z is selected from the group consisting of spermiyl, spermidiyl, aminoacid, peptidyl, diaminoalkyl, and polyamine;

R₇ and R₈ are independently H or a carbohydrate; and

l is an integer from 1 to about 4;

wherein

Q is as defined above, preferably N;

at least one of R₁ and R₄ are straight chain or branched, cyclic, alkyl,alkenyl, alkynyl or aryl groups having from about 8 to about 24 carbonatoms:

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

Z is selected from the group consisting of spermiyl, spermidiyl, aminoacid, peptidyl, diaminoalkyl, and polyamine;

R₇ and R₈ are independently H or a carbohydrate, preferably H;

m and n are as defined above; and

is an integer from 1 to about 4;

wherein

Q, R₁, R₄, r, u, m and n are as defined above:

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

i and j are integers from about 2 to about 3; and

k is an integer from 1 to about 3;

wherein

Q, R₁, R₄, r, u, m and n are as defined above:

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

i and j are integers from about 2 to about 3;

k is an integer from 1 to about 3;

L₁ and L₂, independently from one another, are an alkylene or analkylene ether;

Y is selected from the group consisting of CH₂, O, S and NH;

wherein

Q, R₁, R₄, r, u, m and n are as defined above:

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

i and j are integers from about 2 to about 3;

k is an integer from 1 to about 3;

L₁ and L₂, independently from one another, are an alkylene or analkylene ether; Y is selected from the group consisting of CH₂, O, S andNH;

wherein

Q, R₁, R₄, r, u, m and n are as defined above:

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

i and j are integers from about 2 to about 3;

k is an integer from 1 to about 3;

L₁ and L₂, independently from one another, are an alkylene or analkylene ether;

Y is selected from the group consisting of CH₂, O, S and NH;

wherein

Q, R₁, R₂, r, u, m and n are as defined above;

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

i and j are integers from about 2 to about 3;

k is an integer from 1 to about 3:

L₁ and L₂, independently from one another, are an alkylene or analkylene ether;

Y is selected from the group consisting of CH₂, O, S and NH; and

wherein

Q, R₁, R₄, r, u, m, and n are as defined above;

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

i and j are integers from about 2 to about 3;

k is an integer from 1 to about 3:

L₁ and L₂, independently from one another, are an alkylene or analkylene ether;

Y is selected from the group consisting of CH₂, O, S and NH.

Also, compounds of the present invention have the Formula (B):

wherein

L is (CH₂)_(l) or {(CH₂)_(i)—Y—(CH₂)_(j)}_(k), wherein Y is selectedfrom the group consisting of CH₂, an ether, a polyether, an amide, apolyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, acarbamoyl, a carbonate, and a secondary amino group;

R₁-R₆, independently of one another, are selected from the groupconsisting of H, —(CH₂)_(p)— Z, an alkyl, an alkenyl, an aryl, and analkyl or alkyl ether optionally substituted by one or more of analcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, apolyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or acarbamoyl group, and at least one of R₁, R₃, R₄ and R₆ is a straightchain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group, andanyone or more of R₁, R₄, R₃ and R₆ may optionally be covalently linkedwith each other to form a cyclic moiety;

Z is selected from the group consisting of amine, spermiyl,carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl,pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, andprotein;

A₁ and A₂, independently of one another, are selected from the groupconsisting of CH₂O, CH₂S, CH₂NH, C(O), C(NH), C(S) and (CH₂)_(t);

X is a physiologically acceptable anion;

m, n, v and w are 0 or 1;

i, j, k, l, p and t are integers from 1 to about 100;

q is an integer from 1 to about 1000; and

a is the number of positive charge divided by the valence of the anion,wherein when m and n are 0, then a is 0.

Preferably, R₁-R₆, i, j, k, l, p, t, q, b and c are as defined withreference to Formula (A).

Preferably, Y is selected from the group consisting of CH₂, O, S and NH.

Useful compounds falling within the scope of the Formula (B) includecompounds having the following formulae:

(B1)

wherein

R₁, R₃, R₄ and R₆, independently of one another, are selected from thegroup consisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyloptionally substituted by one or more of an alcohol, an amine, an amide,an ether, a polyether, a polyamide, an ester, mercaptan, a urea, athiourea, a guanidyl, or a carbamoyl group, and at least one of R₁, R₃,R₄ and R₆ is a straight chain or branched, cyclic, alkyl, alkenyl,alkynyl or aryl group having from about 8 to about 24 carbon atoms; and

l, b and c are integers independently selected from 1 to about 4:

wherein

R₁, R₃, R₄ and R₆, independently of one another, are selected from thegroup consisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyloptionally substituted by one or more of an alcohol, an amine, an amide,an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, athiourea, a guanidyl, or a carbamoyl group, and at least one of R₁, R₃,R₄ and R₆ is a straight chain or branched, cyclic, alkyl, alkenyl,alkynyl or aryl group having from about 8 to about 24 carbon atoms;

R₇ and R₈ are independently H or a carbohydrate; and

l is an integer from 1 to about 4;

wherein

R₁, R₂, R₄ and R₅, independently of one another, are selected from thegroup consisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyloptionally substituted by one or more of an alcohol, an amine, an amide,an ether, a polyether, a polyamide, an ester, a mercaptan, a urea, athiourea, a guanidyl, or a carbamoyl group, and at least one of R₁, R₂,R₃ and R₅ is a straight chain or branched, cyclic, alkyl, alkenyl,alkynyl or aryl group having from about 8 to about 24 carbon atoms;

Z is selected from the group consisting of spermiyl, spermidiyl, aminoacid, peptidyl, diaminoalkyl, and polyamine;

m and n are 0 or 1; and

l, b and c are integers independently selected from 1 to about 4;

wherein

at least one of R₁ and R₄ is a straight chain or branched, cyclic,alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24carbon atoms; and

l, b and c are integers independently selected from 1 to about 4;

wherein

at least one of R₁ and R₄ are straight chain or branched, cyclic, alkyl,alkenyl, alkynyl or aryl groups having from about 8 to about 24 carbonatoms; R₇ and R₈ are independently hydrogen or a carbohydrate,preferably hydrogen; and

l is an integer from 1 to about 4;

wherein

Z is as defined above;

at least one of R₁ and R₄ is a straight chain or branched, cyclic,alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24carbon atoms:

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

R₇ and R₈ are independently H or a carbohydrate;

m and n are 0 or 1;

i and j are integers from about 2 to about 3; and

k is an integer from 1 to about 3:

wherein

at least one of R₁ and R₄ is a straight chain or branched, cyclic,alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24carbon atoms;

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁ C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group:

m and n are 0 or 1;

i and j are integers from about 2 to about 3; and

k is an integer from 1 to about 3;

wherein

at least one of R₁ and R₄ is a straight chain or branched, cyclic,alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24carbon atoms;

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl or a carbamoyl group;

m and n are 0 or 1;

i and j are integers from about 2 to about 3; and

k is an integer from 1 to about 3;

wherein

at least one of R₁ and R₄ is a straight chain or branched, cyclic,alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24carbon atoms;

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C1 C8 alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

m and n are 0 or 1;

i and j are integers from about 2 to about 3;

k is an integer from 1 to about 3;

L₁ and L₂, independently from one another, are an alkylene or analkylene ether:

Y is selected from the group consisting of CH₂, O, S and NH;

wherein

at least one of R₁ and R₄ is a straight chain or branched, cyclic,alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24carbon atoms;

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group:

m and n are 0 or 1;

i and j are integers from about 2 to about 3;

k is an integer from 1 to about 3;

L₁ and L₂, independently from one another, are an alkylene or analkylene ether;

Y is selected from the group consisting of CH₂, O, S and NH;

wherein

at least one of R₁ and R₄ is a straight chain or branched, cyclic,alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24carbon atoms;

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁ C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

m and n are 0 or 1;

i and j are integers from about 2 to about 3;

k is an integer from 1 to about 3;

L₁ and L₂, independently from one another, are an alkylene or analkylene ether;

Y is selected from the group consisting of CH₂, O, S and NH; and

wherein

at least one of R₁ and R₄ is a straight chain or branched, cyclic,alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24carbon atoms;

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

m and n are 0 or 1;

i and j are integers from about 2 to about 3;

k is an integer from 1 to about 3:

L₁ and L₂, independently from one another, are an alkylene or analkylene ether;

Y is selected from the group consisting of CHI, O, S and NH; and

wherein

at least one of R₁ and R₅ is a straight chain or branched, cyclic,alkyl, alkenyl, alkynyl or aryl group having from about 8 to about 24carbon atoms;

R₂ and R₅, independently of one another, are selected from the groupconsisting of H and a C₁-C₈ alkyl, alkenyl, aryl, and alkyl optionallysubstituted by one or more of an alcohol, an amine, an amide, an ether,a polyether, a polyamide, an ester, a mercaptan, a urea, a thiourea, aguanidyl, or a carbamoyl group;

m and n are 0 or 1;

i and j are integers from about 2 to about 3;

k is an integer from 1 to about 3;

L₁ and L₂, independently from one another, are an alkylene or analkylene ether;

Y is selected from the group consisting of CH₂, O, S and NH.

In each of formulae (B1) through (B13) preferably R₁ and R₄ are eachC₆₋₃₀ alkyl or alkenyl, more preferably C₈₋₂₄ alkyl or alkenyl, and R₂and R₅ or R₃ and R₆ are each hydrogen or C₁₋₈ alkyl.

Specific compounds within the scope of the invention include thefollowing examples. R₇ and R₈ in the formulae are independently H or acarbohydrate, preferably H.

Further, the compounds according to the present invention have theFormula (C):

wherein

Y is selected from the group consisting of CH₂, an ether, a polyether,an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, aguanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide,an imine a carbonyl, and a secondary amino group and wherein Y isoptionally substituted by —X₁-L′-X₂—Z or —Z;

R₁, R₃, R₄ and R₆, independently of one another, are selected from thegroup consisting of H, —(CH₂)_(p)-D-Z, an alkyl, an alkenyl, an aryl,and an alkyl or an alkyl ether optionally substituted by one or more ofan alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether,a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, aguanidyl, or a carbamoyl group, and at least one of R₁, R₃, R₄ and R₆ isa straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or arylgroup, and R₁, R₃, R₄ and R₆ may optionally be covalently linked witheach other or with Y, to form a cyclic moiety;

Z is selected from the group consisting of amine, spermiyl,carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl,pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, andprotein;

X₁ and X₂, independently of one another, are selected from the groupconsisting of NH, O, S, alkylene, and arylene:

L′ is selected from the group consisting of alkylene, alkenylene,alkynylene, arylene, alkylene ether, and polyether;

D is Q or a bond;

m and n are 0 or 1; and

i, j, k, l and p are integers independently selected from 1 to about 10.

Preferably, Y is selected from the group consisting of CH₂, an ether, apolyether, an amide, a polyamide, an ester, a sulfide, a urea, athiourea, a guanidyl, a carbamoyl, a carbonate, and a secondary aminogroup.

Preferably, R₁, R₃, R₄ and R₆, independently of one another, areselected from the group consisting of H, —(CH₂)_(p)—Z, an alkyl, analkenyl, an aryl, and an alkyl or an alkyl ether optionally substitutedby one or more of an alcohol, an aminoalcohol, an amine, an amide, anether, a polyether, a polyamide, an ester, a mercaptan, a urea, athiourea, a guanidyl, or a carbamoyl group, and at least one of R₁, R₃,R₄ and R₆ is a straight chain or branched, cyclic, alkyl, alkenyl,alkenyl or aryl group, and R₁, R₃, R₄ and R₆ may be covalently linkedwith each other, to form a cyclic moiety.

Preferably, at least one of R₁ and R₄ is straight chain or branched,cyclic, alkyl, alkenyl, alkynyl or aryl group having from about 2 to100, preferably 4 to 75, more preferably 6 to 64, more preferably 8 to50, more preferably 8 to 40, more preferably 8 to 30, more preferably 6to 30, more preferably 4 to 30, and most preferably 8 to about 24 carbonatoms.

Preferably, Y is selected from the group consisting of CH₂, O, S and NH.

The compounds and polycations of the present invention have thefollowing Formula (D):

wherein

L is C, CH, (CH₂), or {(CH₂)_(i)—Y—(CH₂)_(j)}_(k), wherein Y is selectedfrom the group consisting of CH₂, an ether, a polyether, an amide, apolyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, acarbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, acarbonyl, and a secondary amino group and wherein Y is optionallysubstituted by —X₁-L′-X₂—Z or —Z.

R₁-R₆, independently of one another, are selected from the groupconsisting of H, —(CH₂)_(p)-D-Z, an alkyl, an alkenyl, an aryl, and analkyl or an alkyl ether optionally substituted by one or more of analcohol, an amino alcohol, an amine, an amide, an ether, a polyether, apolyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, aguanidyl, or a carbamoyl group, and wherein at least one of R₁, R₃, R₄and R₆ is a straight chain or branched, cyclic, alkyl, alkenyl, alkynylor aryl group;

Z is selected from the group consisting of amine, spermiyl,carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl,pyridyl piperidinyl, pyrrolidinyl, polyamine, amino acid, amino acidderivative, peptide, and protein;

X₁ and X₂, independently of one another, are selected from the groupconsisting of NH, O, S, alkylene and arylene;

L′ is selected from the group consisting of alkylene, alkenylene,alkynylene, arylene, alkylene ether, and polyether;

A₁ and A₂, independently of one another, are selected from the groupconsisting of CH₂O, CH₂S, CH₂NH, C(O), C(NH), C(S) and (CH₂)_(t);

m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both mand n are 0 at least one of r, s, u and y is other than 0:

i, j, k, l, p and t are integers from 0 to about 100; and

q is an integer from 1 to about 1000.

Also, the present invention provides compounds or polycations accordingto the Formula (E):

wherein

L is (CH₂)_(l) or {(CH₂)_(i)—Y— (CH₂)_(j)}_(k), wherein Y is selectedfrom the group consisting of CH₂, an ether, a polyether, an amide, apolyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, acarbamoyl, a carbonate, and a secondary amino group;

R₁-R₆, independently of one another, are selected from the groupconsisting of H, —(CH₂)_(p)—Z, an alkyl, an alkenyl, an aryl, and analkyl or an alkyl ether optionally substituted by one or more of analcohol, an amino alcohol, an amine, an amide, an ether, a polyether, apolyamide, an ester, a mercaptan, a urea, a thiourea, a guanidyl, or acarbamoyl group, and at least one of R₁, R₃, R₄ and R₆ is a straightchain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group; Z, A₁,A₂, m, n, i, j, k, l, p, t and q are as defined above.

In the above formulae (D) and (E), R₁-R₆, Y, i, j, k, l, p, t and q arepreferably as defined with reference to Formula (A).

It would be obvious for a skilled person that when Q is O or S, thenumber of substituents should be according their valency.

Certain of the compounds of the invention may be insufficiently solublein physiological media to employ for delivery and transfection methods.Those of ordinary skill in the art will appreciate that there are avariety of techniques available in the art to enhance solubility of suchcompounds in aqueous media. Such methods are readily applicable withoutundue experimentation to the compounds described herein.

DEFINITIONS

Useful aryl groups are C₆₋₁₀₀ aryl, preferably C₆₋₇₅ aryl, morepreferably C₆₋₆₄ aryl, more preferably C₆₋₅₀ aryl, more preferably C₆₋₄₀aryl, more preferably C₆₋₃₀ aryl, most preferably C₅₋₂₄ aryl. TypicalC₆₋₁₀₀ aryl groups include phenyl, naphthyl, phenanthryl, anthracyl,indenyl, azulenyl, biphenyl, biphenylenyl, fluorenyl, pyrenyl,aceanthrenyl, cholantrenyl, acephenanthrenyl, violantherenyl,hexaphenyl, hexacenyl, trinaphtyl and pyranthyl groups.

Useful alkyl groups are straight chain or branched C₂₋₁₀₀ alkyl groups,preferably C₄₋₇₅ alkyl, more preferably C₆₋₆₄ alkyl, more preferablyC₈₋₅₀ alkyl, more preferably C₈₋₄₀ alkyl, more preferably C₈₋₃₀ alkyl,more preferably C₆₋₃₀ alkyl, more preferably C₄₋₃₀ alkyl, mostpreferably C₈₋₂₄ alkyl. Typical C₂₋₁₀₀ alkyl groups include ethyl,propyl, isopropyl, butyl, sec.-butyl, tert.-butyl, pentyl, hexyl, octyl,decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, eicosyl, docosyl,tetracosyl, hexacosyl, octacosyl and triacontyl groups. Alsocontemplated is a trimethylene group substituted on two adjoiningpositions on any benzene ring of the compounds of the invention.

Useful alkenyl groups are straight chain or branched C₂₋₁₀₀ alkenyl,preferably C₄₋₇₅ alkenyl, more preferably C₆₋₆₄ alkenyl, more preferablyC₈₋₅₀ alkenyl, more preferably C₈₋₄₀ alkenyl, more preferably C₈₋₃₀alkenyl, more preferably C₆₋₃₀ alkenyl, more preferably C₄₋₃₀ alkenyl,most preferably C₈₋₂₄ alkenyl. Typical C₂₋₁₀₀ alkenyl groups includeethenyl, propenyl, isopropenyl, butenyl, sec.-butenyl, hexenyl, octenyl,decenyl, dodecenyl, especially 9-dodecenyl, tetradecenyl, especially9-tetradecenyl, hexadecenyl, especially 9-hexadecenyl, octadecenyl,especially 9-octadecenyl, eicosenyl, docosenyl, tetracosenyl,hexacosenyl, octacosenyl and triacontenyl.

Useful alkynyl groups are straight chain or branched C₂₋₁₀₀ alkynyl,preferably C₄₋₇₅ alkynyl, more preferably C₆₋₆₄ alkynyl, more preferablyC₈₋₅₀ alkynyl, more preferably C₈₋₄ alkynyl, more preferably C₈₋₃₀alkynyl, more preferably C₆₋₃₀ alkynyl, more preferably C₄₋₃₀ alkynyl,most preferably C₈₋₂₄ alkynyl. Typical C₂₋₁₀₀ alkynyl groups includeethynyl, propynyl, butynyl, -butynyl, hexynyl, octynyl, decynyl,dodecynyl, tetradecynyl, hexadecynyl, octadecynyl, eicosynyl, docosynyl,tetracosynyl, hexacosynyl, octacosynyl and triacontynyl groups.

Typical alkyl ether groups include any of the above-mentioned C₂₋₁₀₀alkyl groups having an ether group.

An ether group is —O—.

Typical polyether groups include the —(CHR¹⁴—CH₂—O)_(t)—, wherein R¹⁴ isH or a C₁₋₄ alkyl group and t is an integer as defined above, preferablyt is 2 to 5.

For the purposes of the invention an amide group is an organic radicalhaving —NHC(O)— as a functional group. Typical amide groups includealkyl amides, alkenyl amides, alkynyl amides, and aryl amides, whereinalkyl, alkenyl, alkynyl and aryl are as defined above.

Typically polyamide groups include organic radicals having two or moreamide groups as defined above.

Typically an ester group is an organic radical having —C(O)—O— as afunctional group. Typical ester groups include R¹⁴—C(O)—O—R¹⁵, whereinR¹⁴ and R¹⁵ are alkylene, alkenylene, alkynylene and arylene groups asdefined above.

Typically urea groups are organic radicals having —NH—C(O)—NH— as afunctional group. Typical urea groups include R¹⁴NH—C(O)—NHR¹⁴,R¹⁴NH—C(O)—NHR¹⁵, R¹⁴R¹⁵N—C(O)—NR¹⁴R¹⁵ wherein R¹⁴ and R¹⁵ are alkylene,alkenylene, alkynylene and arylene groups as defined above.

Typically thiourea groups are organic radicals having urea group asdefined above wherein the oxygen in the urea group is substituted bysulfur.

Typically guanidyl groups are organic radicals having —NH—C(NH)—NH— as afunctional group. Typical guanidyl groups include R¹⁴NH—C(NH)—NHR¹⁴,R¹⁴NH—C(NH)—NHR¹⁵ and R¹⁴R¹⁵N—C(NH)—NR¹⁴R¹⁵ wherein R¹⁴ and R¹⁵ arealkylene, alkenylene, alkynylene and arylene groups as defined above.

A carbamoyl group is —NH—C(O)—O—.

Typically carbonate groups include organic radicals containing a CO₃ ²—radical, i.e., —O—C(O)—O.

A phosphate group is a PO₄ ³— radical.

A sulfate group is a SO₄ ²— radical.

A sulfoxide group is —S(O)—.

An imine group is —C(N)—.

A carbonyl group is —C(O)—.

A secondary amino group is —NH—.

Typically amino alcohol groups are organic radicals having both asecondary amino group as defined above and a hydroxyl group. Typicalaminoalcohols include amino ethanol, aminopropanol and aminobutanol.

The definition “D is a bond” means that when D is not Q there is asingle bond between (CH₂)_(p) and Z.

Biologically Active Substance refers to any molecule or mixture orcomplex of molecules that exerts a biological effect in vitro and/or invivo, including pharmaceuticals, drugs, proteins, peptides,polypeptides, hormones, vitamins, steroids, polyanions, nucleosides,nucleotides, nucleic acids (e.g. DNA or RNA), nucleotides,polynucleotides, etc.

Cationic Lipids refers to any cationic lipids which may be used fortransfection, including but not limited to, DOSPA, DOTMA, DMRIE, DOTAP,DOGS and TM-TPS.

Cell refers to eukaryotic cells of any type and from any source. Typesof eukaryotic cells include epithelial, fibroblastic, neuronal,hematopoietic cells and the like from primary cells, tumor cells orimmortalized cell lines. Sources of such cells include any animal suchas human, canine, mouse, hamster, cat, bovine, porcine, monkey, ape,sheep, fish, insect, fungus and any plant including crop plants,ornamentals and trees.

Delivery is used to denote a process by which a desired compound istransferred to a target cell such that the desired compound isultimately located inside the target cell or in, or on, the target cellmembrane. In many uses of the compounds of the invention, the desiredcompound is not readily taken up by the target cell and delivery vialipid aggregates is a means for getting the desired compound into thecell. In certain uses, especially under in vivo conditions, delivery toa specific target cell type is preferable and can be facilitated bycompounds of the invention.

Drug refers to any therapeutic or prophylactic agent other than foodwhich is used in the prevention, diagnosis, alleviation, treatment, orcure of disease in man or animal.

Kit refers to transfection or protein expression kits which include oneor more of the compounds of the present invention or mixtures thereof.Such kits may comprise a carrying means being compartmentalized toreceive in close confinement one or more container means such as vials,test tubes and the like. Each of such container means comprisescomponents or a mixture of components needed to perform transfection.Such kits may include one or more components selected from nucleic acids(preferably one or more vectors), cells, one or more compounds of thepresent invention, lipid-aggregate forming compounds, transfectionenhancers, biologically active substances, etc.

Lipid Aggregate is a generic term which includes liposomes of all typesboth unilamellar and multilameller as well as micelles and moreamorphous aggregates of cationic lipids or lipids mixed with amphiphaticlipids such as phospholipids and steroids.

Lipid Aggregate-forming Compounds refers to neutral compounds or lipidssuch as DOPE, DOPC and cholesterol, etc.

Target Cell refers to any cell to which a desired compound is delivered,using a lipid aggregate as carrier for the desired compound.

Transfection is used herein to mean the delivery of nucleic acid,protein or other macromolecule to a target cell, such that the nucleicacid, protein or other macromolecule is expressed or has a biologicalfunction in the cell. The term “expressible nucleic acid” includes bothDNA and RNA without regard to molecular weight, and the term“expression” means any manifestation of the functional presence of thenucleic acid within the cell including, without limitation, bothtransient expression and stable expression. Functional aspects includeinhibition of expression by oligonucleotides or protein delivery.

Transfection Enhancers refers generally to molecules and substances suchas proteins, peptides, growth factors and the like that enhancecell-targeting, uptake, internalization, nuclear targeting andexpression. Such molecules and substances include ligands such asinsulin, transferrin, fibronectin that target the cell surface; peptidesthat target cellular integrin receptors; and other compounds such asPlus Reagent (available from Life Technologies, Inc., Rockville, Md.).Examples of transfection enhancers may be found in U.S. Pat. No.5,736,392 and U.S. application Ser. No. 09/039,780 filed Mar. 16, 1998.

The invention will be further clarified by the following examples, whichare intended to be purely exemplary of the invention. The polycationiclipids were prepared by following the general reaction schemes describedbelow.

EXAMPLES Example 1 Synthesis of N₁,N⁴-dioleoyl-diaminobutane (I)

A solution of 1,4-diaminobutane (4.28 g, 48.6 mmol) and triethylamine(20.4 ml, 146 mmol) in 10 mL of dry methylene chloride was slowly addedto a solution of oleoyl chloride (30.0 g, 99.7 mmol) in 300 ml ofanhydrous methylene chloride in an ice bath 25 at 0° C. The reactionmixture was stirred vigorously with a mechanical stirrer. After theaddition was complete, the ice bath was removed and the mixture wasstirred at room temperature for 2.5 days. TLC analysis confirmed thatthe reaction had gone to completion and the product had precipitated.The excess oleoyl chloride was removed by filtration. The precipitatewas washed twice with 50 ml of methylene chloride. The mother liquor wasconcentrated and more product precipitated. This precipitate wasfiltered and combined with the previous precipitate. The resulting solidwas vacuum dried for 4 hours. A total of 27.0 g of a white solid of thedesired product, N¹,N⁴-dioleoyl-diaminobutane, was obtained.

Synthesis of N1,N-dioleyl-diaminobutane (II)

Lithium aluminum hydride (8.62 g, 95%, 216 mmol) was carefully added toa suspension of N¹,N⁴-dioleoyl-diaminobutane (27.0 g, 43.8 mmol) in 400ml of anhydrous diethyl ether at 0° C. After addition, the ice bath wasremoved. The reaction mixture was warmed slowly to room temperature andthen heated gently to reflux with an appropriate condensing device andstirred for 16 hours. The reaction mixture was then cooled and quenchedcarefully at 0° C. with 70 mL of a 1 N sodium hydroxide solution.Another 500 mL of diethyl ether was added and the mixture was stirred atroom temperature for additional 2 hours. The top ether layer turnedclear gradually and then separated. The aqueous layer was extractedthree times with 100 mL of diethyl ether each. The combined ethersolution was concentrated, and dried on high vacuum overnight. Total of17.0 g of oily colorless N¹,N⁴-dioleyl-diaminobutane was obtained.

Synthesis ofN¹,N⁴-dioleyl-N¹,N⁴-di-[2-hydroxy-3-(N-phthalamido)propyldiamino-butane(III)

Diisopropylethylamine (11.1 mL, 63.7 mmol) was added to a suspension ofN¹,N⁴-dioleyl-diaminobutane (15.5 g, 26.3 mmol) andN-(2,3-epoxypropyl)-phthalimide (15.6 g, 76.8 mmol) in 110 mL of dryN,N-dimethylformamide. After purging with nitrogen, the reaction mixturewas sealed in a round-bottom flask and heated to around 90° C. for 24hours. N,N-dimethylformamide and diisopropylethylamine were removed anda yellow oil was obtained. This crude material was recrystallized fromethanol. A total of 18.6 g of a white solid,N¹,N⁴-dioleyl-N¹,N⁴-di-[2-hydroxy-3-(N-phthalamido)propyl]-diamino-butanewas obtained.

Synthesis ofN¹,N⁴-dioleyl-N¹,N⁴-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane (IV)(hereinafter referred to as DHDOS)

Hydrazine (4.0 mL, 80% aq., 103 mmol) was added to a suspension ofN¹,N⁴-dioleyl-N¹,N⁴-di-[2-hydroxy-3-(N-phthalamido)propyl]-diaminobutane(17.0 g, 17.1 mmol) in 250 mL of dry ethanol at room temperature. Withan appropriate condensing device, the reaction mixture was heated to areflux, at which point the suspension turned into a clear solution. Theoil bath was set to 85° C. After 45 minutes a white solid precipitatedfrom the solution. The reaction mixture was stirred at reflux for 4hours before being cooled to −20° C. The white solid settled down to thebottom. The top clear ethanol solution was decanted. The residue waswashed twice with cold ethanol. The combined ethanol solution wasconcentrated and dried overnight over vacuum. 12.4 g of oilyN¹,N⁴-dioleyl-N¹,N⁴-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane wasobtained.

The following compounds were synthesized by the above method using thecorresponding diamine and a long chain acyl chloride:

-   N¹,N⁴-dimyristyl-N¹,N⁴-di-[2-hydroxy-3-(N-aminopropyl-)]-diaminobutane;-   N¹,N⁴-dipalmityl-N¹,N⁴-di-[2-hydroxy-3-(N-aminopropyl-)]-diaminobutane;-   N¹,N⁴-dipalmitolyl-N    N¹,N⁴-di-[2-hydroxy-3-(N-aminoprop-yl)]-diaminobutane;-   N¹,N⁴-distearyl-N¹,N⁴-di-[2-hydroxy-3-(N-aminopropyl)-]-diaminobutane;-   N¹,N⁴-dilauryl-N¹,N⁴-di-[2-hydroxy-3-(N-aminopropyl)]-diaminobutane;-   N¹,N²-dimyristyl-N¹,N²-di-[2-hydroxy-3-(N-aminopropyl-)]-diaminoethane;-   N¹,N²-dipalmity-N¹,N²-di-[2-hydroxy-3-(N-aminopropyl)-]-diaminoethane;-   N¹,N²-dipalmitolyl-N¹,N²-di-[2-hydroxy-3-(N-aminoprop-yl)]-diaminoethane;-   N¹,N²-distearyl-N¹,N²-di-[2-hydroxy-3-(N-aminopropyl)-]-diaminoethane;-   N¹,N²-dilauryl-N¹,N²-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;-   N¹,N²dioleyl-N¹,N²-di-[2-hydroxy-3-(N-aminopropyl)]-diaminoethane;-   N¹,N⁸-dimyristyl-N¹,N⁸-di-[2-hydroxy-3-(N-aminopropyl-)]-Jeffamine;-   N¹,N⁸-dipalmityl-N¹,N⁸-di-[2-hydroxy-3-(N-aminopropyl-)]-Jeffamine;-   N¹,N⁸-dipalmitolyl-N¹,N⁸-di-[2-hydroxy-3-(N-aminoprop-yl)]-Jeffamine;-   N¹,N⁸-distearyl-N¹,N⁸-di-[2-hydroxy-3-(N-aminopropyl)-]-Jeffamine;-   N¹,N⁸-dilauryl-N¹,N⁸-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;-   N¹,N⁸-dioleyl-N¹,N⁸-di-[2-hydroxy-3-(N-aminopropyl)]-Jeffamine;

Synthesis ofN¹,N⁴-dioleyl-N¹,N⁴-di-[2-hydroxy-3-(N-carboxamidine)-aminopropyl]-diaminobutane(V)

1H-pyrazole-1-carboxamidine hydrochloride (45 mg, 0.31 mmol) was addedto a solution ofN¹,N⁴-dioleyl-N¹,N⁴-di-[2-hydroxy-3-(N-aminopropyl)]-diamino-butane (115mg, 0.156 mmol) in 1 mL of dry N,N-dimethylformamide. The salt was notvery soluble in dimethylformamide (DMF). However, the mixture turnedclear after diisopropylethylamine (55 μl, 0.31 mmol) was added. Themixture was stirred under nitrogen at room temperature for 18 hours.After removal of solvent, the crude material was loaded on a C-18reverse phase flash column, and eluted with 20% H₂O in MeOH to 10% H₂Oin MeOH. The pure fractions were collected and concentrated. An 81 mgcolorless oilyN¹,N⁴-dioleyl-N¹,N⁴-di-[2-hydroxy-3-(N-carboxamidine)-aminopropyl]-diaminobutanewas obtained, which was converted to its TFA and HCL salts.

Synthesis ofN¹,N⁴-dioleyl-N¹,N⁴-di-{2-hydroxy-3-[N(N^(I),N^(II),N^(III),N^(IV)-butoxycarbonyl-sperminecarboxamido)]aminopropyl}diaminobutane (VI)

Diisopropylcarbodiimide (5.32 mL, 34.0 mmol) was added drop wise to asolution of Boc-spermine acid (21.7 g, 33.5 mmol) andN-hydroxysuccinimide (NHS) (3.91 g, 34.0 mmol) in mixed solvents (100 mLof DMF and 100 mL of CH₂Cl₂) at room temperature. After stirring for 2.5hours, a solution ofN¹,N⁴-dioleyl-N¹,N⁴-di-[2-hydroxy-3-(N-aminopropyl)]diaminobutane (10 g,13.6 mmol) in 40 mL of methylene chloride and DMF was added. The mixturewas stirred for another 5 hours before quenching with 200 mL of a 2.5%sodium bicarbonate solution. An additional 300 mL of methylene chloridewas added. The aqueous solution was extracted with 120 mL of methylenechloride three times. The combined organic solution was washed withwater twice and dried over anhydrous magnesium sulfate. Afterconcentration, a pale yellow oil was obtained. The crude material waspurified with silica gel, eluting with 2% MeOH in CH₂Cl₂ to 5% MeOH inCH₂Cl₂. A total of 13.1 g of white solidN¹,N⁴-dioleyl-N¹,N⁴-di-{2-hydroxy-3-[N—(N^(I),N.sup-.^(II),N^(III),N^(IV)-butoxycarbonyl-sperminecarboxamido)]aminopropyl}diaminobutane was obtained.

Synthesis of N¹,N⁴-dioleyl-N¹,N⁴-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminobutane (VII)

100 mL of a solution of 4.0 M hydrogen chloride in 1,4-dioxane was addedto a solution ofN¹,N⁴-dioleyl-N¹,N⁴-di-{2-hydroxy-3-[N—(N^(I),N^(II),N^(III),N^(IV)butoxycarbonylspermine carboxamido)]aminopropyl}diaminobutane (11.8 g,5.92 mmol) in 100 mL of 1,4-dioxane at room temperature. The reactionmixture turned cloudy 10 minutes after addition of the acid. After 2.5hours of stirring at room temperature, the excess acid and solvent wasremoved. The residue was dried for at least 5 hours over vacuum beforebeing loaded on a C-18 reverse phase flash column. The column was elutedstarting with 25% H₂O in MeOH, then 20%, and then 17%. Pure fractionswere collected and concentrated. A 3.06 g colorless solidN¹,N⁴-dioleyl-N¹,N⁴-di-[2-hydroxy-3-(N-spenninecarbox-amido)-aminopropyl]-diaminobutanewas obtained.

The following compounds were synthesized using the protocol describedabove starting with the requisite diamine and long chain acyl chloride:

-   N¹,N⁴-dimyristyl-N¹,N⁴-di-[2-hydroxy-3-(N-sperminecar-boxamido)-aminopropyl]-diaminobutane;-   N¹,N⁴-dipalmityl-N¹,N⁴-di-[2-hydroxy-3-(N-sperminecar-boxamido)-aminopropyl]-diaminobutane;-   N¹,N⁴-dipalmitolyl-N¹,N⁴-di-[2-hydroxy-3-(N-sperminec-arboxamido)-aminopropyl]-diaminobutane;-   N¹,N⁴-distearyl-N¹,N⁴-di-[2-hydroxy-3-(N-sperminecarb-oxamido)-aminopropyl]-diaminobutane;-   N¹,N⁴-dilauryl-N¹,N⁴-di-[2-hydroxy-3-(N-sperminecarbo-xamido)-aminopropyl]-diaminobutane;-   N¹,N⁸-dimyristyl-N¹,N⁸-di-[2-hydroxy-3-(N-sperminecar-boxamido)-aminopropyl]-Jeffamine;-   N¹,N⁸-dipalmityl-N¹,N⁸-di-[2-hydroxy-3-(N-sperminecar-boxamido)-aminopropyl]-Jeffamine;-   N¹,N⁸-dipalmitolyl-N¹,N⁸-di-[2-hydroxy-3-(N-sperminec-arboxamido)-aminopropyl]-Jeffamine;-   N¹,N⁸-distearyl-N¹,N⁸-di-[2-hydroxy-3-(N-sperminecarb-oxamido)-aminopropyl]-Jeffamine;-   N¹,N⁸-dilauryl-N¹,N⁸-di-[2-hydroxy-3-(N-sperminecarbo-xamido)-aminopropyl]-Jeffamine;-   N¹,N⁸-dioleyl-N¹,N⁸-di-[2-hydroxy-3-(N-sperminecarbox-amido)-aminopropyl]-Jeffamine;-   N¹,N²-dimyristyl-N¹,N^(Z)-di-[2-hydroxy-3-(N-sperminecar-boxamido)-aminopropyl]-diaminoethane;-   N¹,N²-dipalmityl-N¹,N²-di-[2-hydroxy-3-(N-sperminecar-boxamido)-aminopropyl]-diaminoethane;-   N¹,N²-dipalmitolyl-N¹,N²-di-[2-hydroxy-3-(N-sperminec-arboxamido)-aminopropyl]-diaminoethane;-   N₁,N²-distearyl-N¹,N²-di-[2-hydroxy-3-(N-sperminecarb-oxamido)-aminopropyl]-diaminoethane;-   N¹,N²-dilauryl-N¹,N²-di-[2-hydroxy-3-(N-sperminecarbo-xamido)-aminopropyl]-diaminoethane;-   N¹,N²-dioleyl-N¹,N²-di-[2-hydroxy-3-(N-sperminecarbox-amido)-aminopropyl]-diaminoethane;

Example 2 Synthesis of N¹,N⁴-dioleyl-N¹,N⁴-di-3-cyanopropyldiaminobutane(VIII)

Acrylonitrile (0.43 mL, 6.53 mmol) was added dropwise to a solution ofN¹,N⁴-dioleyl-diaminobutane (1.8 g, 3.06 mmol) in 20 mL of ethanol atroom temperature. The mixture was stirred for 30 hours. All startingmaterials were converted to product as confirmed by TLC analysis. Thecrude material was purified using flash chromatography with a silica gel(1% MeOH in CH₂Cl². A clear oil was obtained at high yield.

Synthesis of N¹,N₄-dioleyl-N¹,N⁴-di-3-(aminopropyl)-diaminobutane (XI)

A solution of LAH (9.2 mL, 1 M in ether, 9.2 mmol) was slowly added to asolution of N¹,N⁴-dioleyl-N¹,N⁴-di-3-cyanopropyl-diaminobutane (2.12 g,3.05 mmol) in 15 mL of anhydrous diethyl ether at 0° C. After addition,the mixture was stirred at room temperature for 20 hours. All startingmaterial was consumed. The reaction mixture was quenched with a 1 N NaOHsolution at 0° C. After stirring 2 hours at room temperature, themixture was extracted with diethyl ether three times. The combined ethersolutions were concentrated and dried over vacuum for three hours. Anoily N¹,N⁴-dioleyl-N¹,N⁴-di-3-(aminopropyl)diaminobutane was obtained athigh yield.

Synthesis of N¹,N⁴-dioleyl-N¹,N⁴-di-[3-(N-sperminecarboxamido)-aminopropyl]-diaminobutane (XI)

The procedure for making N¹,N⁴-dioleyl-N¹,N⁴-di-[2-hydroxy-3-(N-sperminecarboxamido)-aminopropyl]-diaminobutane described above was followed.

Example 3 Synthesis of Cholesterol Analogs

The cholesterol analogs can be synthesized by using the scheme givenbelow (Scheme 3). Jeffamine is alkylated with cholestryl chloride toprovide the dicholestryl jeffamine analog (XII). Further alkylation withthe epoxide phthalamide (XIII) and deblocking with hydrazine gives thecompound of the invention (XIV).

Example 4 Synthesis of Monoalkyl Analogs

When monoalkyl analogs are desired, the above Scheme 1 can be modifiedsuch that one of the amines in the starting material is protected beforethe acylation step. Thus, tritylprotected diaminobutane (XV) is acylatedwith alkanoyl chloride (e.g., oleoyl chloride) followed with LAHreduction to obtain compound XVIII. The amine is then alkylated with thedesired phthalamide epoxide to obtain compound XVIII. Removing thephthalamide using hydrazine renders the desired amine XIX. (See Scheme4).

Example 5 Synthesis of Cyclic Analogs

The following scheme (Scheme 5) can be used to make the cyclic analogs.Trityl protected amino alcohol (XX) with the desired chain is alkylatedusing dibromoalkyl (e.g., dibromobutane). The trityl is removed from thedesired dimer (XXI) and acylated using diacyl chlorides (e.g., succinylchloride). The amide (XXIII) is then reduced with LAB and alkylatedusing the desired phthalamide epoxide. Removal of the phthalamide givesthe desired compound of the invention.

Example 6 Synthesis of Polymeric Analogs

Polymeric analogs of the present invention can be synthesized by usingpolymeric amines such as PEI as starting material or dendrimericpolyamines. For example, PEI can be acylated with alkyloyl chloride(e.g., oleoyl chloride) and the acylated PEI can then be reduced withlithium aluminum hydride to obtain compounds of the invention.

Although the above methods exemplify the synthesis of specificcompounds, the reaction schemes provide a general method for preparing avariety of compounds according to the present invention. Those ofordinary skill in the art will appreciate that alternate methods andreagents other than those specifically detailed herein can be employedor readily adapted to produce compounds of the invention.

The compounds of the present invention can be used in the same manner asare prior art compounds such as DOTMA, DOTAP, DOGS, DOSPA and the like.Methods for incorporating such cationic lipids into lipid aggregates arewell-known in the art. Representative methods are disclosed by Felgner,et al., supra; Eppstein et al. supra; Behr et al. supra; Bangham, A. etal. (1965) M. Mol. Biol. 23:238 252; Olson, F. et al. (1979) Biochim.Biophys. Acta 557:9 23; Szoka, F. et al. (1978) Proc. Natl. Acad. Sci.USA 75:4194 4198; Mayhew, E. et al. (1984) Biochim. Biophys. Acta775:169 175; Kim, S. et al. (1983) Biochim. Biophys. Acta 728: 339 348;and Fukunaga, M. et al. (1984) Endocrinol. 115:757 761. Techniques forpreparing lipid aggregates of appropriate size for use as deliveryvehicles include sonication and freeze-thaw plus extrusion as perhapsthe most commonly used. See, e.g., Mayer, L. et al. (1986) Biochim.Biophys. Acta 858:161 168. Microfluidization is used when consistentlysmall (50 200 nm) and relatively uniform aggregates are desired (Mayhew,E., supra). Aggregates ranging from about 50 nm to about 200 nm diameterare preferred; however, both larger and smaller sized aggregates arefunctional.

Methods of transfection and delivery of other compounds are well-knownin the art. The compounds and compositions of the present inventionyield lipid aggregates that can be used in the same processes used forother known transfection agents.

It will be readily apparent to those of ordinary skill in the art that anumber of general parameters are important for optimal efficiency oftransfection or delivery. These parameters include, for example, thecationic lipid concentration, the concentration of compound to bedelivered, the number of cells transfected, the medium employed fordelivery, the length of time the cells are incubated with thepolyanion-lipid complex, and the relative amounts of cationic andnon-cationic lipid. It may be necessary to optimize these parameters foreach particular cell type. Such optimization is routine employing theguidance provided herein and knowledge generally available to the art.

It will also be apparent to those of ordinary skill in the art thatalternative methods, reagents, procedures and techniques other thanthose specifically detailed herein can be employed or readily adapted toproduce the liposomal precursors and transfection compositions of thisinvention. Such alternative methods, reagents, procedures and techniquesare within the spirit and scope of this invention.

The use of representative compounds of the invention are furtherdetailed by reference to the following examples. All abbreviations usedherein are standard abbreviations in the art. Specific procedures notdescribed in detail are either referenced or well-known in the art.

Example 7

This example compares transfection of HEK-293 (human embryonickidney-derived cell line), COS-7 (SV40 transformed monkey cell line),CHO-KI (Chinese Hamster Ovary-derived cell line), and HeLa (Humancervical carcinoma-derived cell line) cells with the β-galactosidasereporter plasmid DNA pCMV SPORT-β-gal (Life Technologies, Rockville,Md.) using commercially available cationic lipid transfection reagentsand the compounds of the present invention.

The cells were plated the day before transfection in 24-well tissueculture plates in a total volume of 0.4 ml DMEM (Dulbecco's ModifiedEagle's Medium, Life Technologies, Rockville, Md.) culture mediumcontaining a 1% non-essential amino acid (NEAA) solution(LifeTechnologies), and 10% FBS. For the HEK-293 and COS-7 cells, tissueculture plates were pre-coated with Poly-L-Lysine to enhance cellattachment.

The next day, DNA-transfection reagent complexes were prepared asfollows:

The cationic lipid reagents and DNA were diluted separately into 25.mu.laliquots of serum-free DMEM, containing 1% NEAA. For LIPOFECTAMINE®PLUS, 7 14 μl of PLUS reagent was added to the DNA, mixed, and incubatedfor 15 minutes at room temperature. The diluted DNA was combined withthe diluted lipid and incubated at room temperature for at least 15minutes to allow the DNA and the lipid to form complexes. Following thisincubation the complexes were added directly into the culture mediumdropwise and mixed by rocking the culture plate back and forth. Thecells were further incubated at 37° C. for a total of 24 hours to allowexpression of the lacZ transgene encoded by the reporter plasmid, pCMVSPORT-β-gal. At 24 hours post-transfection, the growth medium andtransfection complexes were removed from the wells, and the cells ineach well were rinsed briefly with 1 ml of D-PBS (Dulbecco's PBS, LifeTechnologies, Rockville, Md.). The cells in each well were lysed by theaddition of 0.15 to 2.0 ml of 0.1% Tris, pH 8.0, containing 0.1 M TritonX-100. The plates were frozen at −80° C. for a minimum of 2 hours, andthawed at room temperature or 37° C. The thawed cell lysates werecleared by centrifugation and the supernatants were assayed for β-galactivity using the enzymatic substrate ONPG. The concentration of totalprotein in cell lysates was also determined using a Bradford assay(Bio-Rad Laboratories, Hercules Calif.). β-gal activity in transfectedcell extracts was calculated against a standard curve and expressed asng β-gal per surface area of tissue culture plate (ng/cm2) to reflectactivity per transfection, or as ng β-gal per μg of total protein(ng/μg) to reflect specific activity.

HEK-293 (FIG. 1), COS-7 (FIG. 2), CHO-KI (FIG. 3), and HeLa (FIG. 4)cells were transfected with 0.4 or 0.8 μg of pCMV SPORT-β-gal DNA and0.2 to 4.0 μl of transfection reagent. The transfection reagents testedwere DHDOS (IV) formulated at 2 mg/ml with the neutral co-lipid,cholesterol (at a ratio of 1:15 (M/M) DHDOS to cholesterol); DHDOSformulated at 2 mg/ml with the neutral co-lipid DOPE(dioleylphosphatidyl ethanolamine) (at a ratio of 1:1 (M/M) DHDOS toDOPE); LIPOFECTAMINE® PLUS (Life Technologies, Rockville Md.); andFuGENE™-6 (Boehringer Mannheim, Germany). DHDOS formulations were testedin the range of 0.2 to 1.5 μl; LIPOFECTAMINE® PLUS and FuGENE-6 weretested in the range of 0.2 to 4.0 μl. FuGENE-6 was used according to themanufacturer's recommended protocol. DHDOS and LIPOFECTAMINE® PLUS wereused according to the above protocol. The data presented in the Figuresare expressed as total activity (ng/cm²) to better compare totalexpression from the transfected DNA. Only data with 0.8 μg of DNA isshown, since similar results were obtained with 0.4 and 0.8 μg of DNA.

Example 8

Primary, passaged, normal human fibroblasts (NHFs) were plated in96-well plates at a density of 1.6.times.104 cells per well andtransfected the following day. Cells in each well were transfected with40 ng pCMV SPORT-β-gal DNA and 0.1 or 0.2 μl lipid.

The DNA and lipid were diluted separately into 10 μl of DMEM. The DNAwas either used alone or pre-mixed with PLUS, insulin, transferrin, oran integrin-targeting peptide prior to complexing with the lipid. After15 minutes of complexing, the DNA-lipid was added to cells. Cells wereassayed for p-gal activity as described above.

ACTIVITY (ng/βgal/cm²) DNA and INTEGRIN- DNA and DNA and TARGETING LIPIDDNA DNA and PLUS INSULIN TRANSFERRIN PRPTIDE LipofectAMINE 10.36 28.6 ND17.4 ND Compound of ND 37.5 ND ND  40.9 Formula X 1:1.5 DOPE 1 mg/mlCompound of 29.4  637.9 195.7 21.7 587.9 Formula VII 2 mg/ml ND = nodetectable activity *PLUS Reagent is available from Life Technologies,Inc., Rockville, Maryland **Reference: S.L. HART, et al (1998), HumanGene Therapy, 9: 575-585

The results show that these cationic lipid formulations can deliver DNAmolecules alone, but also that delivery, and ultimately gene expression,may be enhanced when the lipids are used in conjunction with peptides orproteins that bind DNA and/or act as ligands for cell surface receptors,or otherwise enhance cellular and/or nuclear uptake.

Having now fully described the present invention in some detail by wayof illustration and examples for purposes of clarity of understanding,it will be obvious to one of ordinary skill in the art that the same canbe performed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1-20. (canceled)
 21. A transfection complex formed in an aqueous medium,said transfection compex comprising: an expressible nucleic acid; acompound having the structure:

 or a polycation thereof; wherein R₄ is a straight-chain, branched orcyclic, alkyl or alkenyl group having 8 to 24 carbon atoms, R₅ is analkyl group substituted with one or more amines and one or morealcohols, and R₇ is H or a carbohydrate; and at least one protein orpeptide transfection enhancer.
 22. The transfection complex according toclaim 21, wherein the protein or peptide transfection enhancer enhancescell-targeting, uptake, internalization, nuclear targeting or expressionof the expressible nucleic acid.
 23. The transfection complex accordingto claim 21, wherein the protein or peptide transfection enhances uptakeof the expressible nucleic acid.
 24. The transfection complex accordingto claim 21, wherein R₅ is an alkyl group substituted with one amine andone or more alcohols.
 25. The transfection complex according to claim21, wherein the amine substituent of the R₅ alkyl group comprises anaminoethanol, an aminopropanol or an aminobutanol group.
 26. Thetransfection complex according to claim 2524, wherein the aminesubstituent of the R₅ alkyl group has the structure:

wherein n is an integer from 7-23.
 27. The transfection complexaccording to claim Error! Reference source not found., wherein R₅ is analkyl group having 3-30 carbon atoms and is substituted by one amine andone or more alcohols.
 28. The transfection complex according to claim27, wherein the amine substituent of the R₅ alkyl group comprises anaminoethanol, an aminopropanol or an aminobutanol group.
 29. Thetransfection complex according to claim 28, wherein the aminesubstituent of the R₅ alkyl group has the structure:

wherein n is an integer from 7-23.
 30. The transfection complexaccording to claim 29, wherein the amine substituent of the R₅ alkylgroup comprises an aminoethanol, an aminopropanol or an aminobutanolgroup.
 31. The transfection complex according to claim 30, wherein theamine substituent of the R₅ alkyl group has the structure:

wherein n is an integer from 7-23.
 32. The transfection complexaccording to claim Error! Reference source not found., wherein R₇ is H.33. The transfection complex according to claim 32, wherein R₄ is astraight-chain alkyl group having 8 to 24 carbon atoms.
 34. Thetransfection complex according to claim 33, wherein R₅ is an alkyl grouphaving 3-30 carbon atoms and is substituted with one amine and one ormore alcohols.
 35. The transfection complex according to claim 34,wherein the amine substituent of the R₅ alkyl group comprises anaminoethanol, an aminopropanol or an aminobutanol group.
 36. Thetransfection complex according to claim 34, wherein the aminesubstituent of the R₅ alkyl group has the structure:

wherein n is an integer from 7-23.
 37. The transfection complexaccording to claim 34, wherein R₅ is a propyl or a butyl group which issubstituted by one amine and one or more alcohols.
 38. The transfectioncomplex according to claim 37, wherein the amine substituent of the R₅alkyl group comprises an aminoethanol, an aminopropanol or anaminobutanol group.
 39. The transfection complex according to claim 38,wherein the amine substituent of the R₅ alkyl group has the structure:

wherein n is an integer from 7-23.
 40. The transfection complexaccording to claim Error! Reference source not found., wherein R₄ is astraight-chain alkyl group having 8 to 24 carbon atoms, R₅ is a butylgroup substituted with one amine and one or more alcohols, and R₇ is H.41. The transfection complex according to claim 40, wherein the aminesubstituent of the R₅ alkyl group comprises an aminoethanol, anaminopropanol or an aminobutanol group.
 42. The transfection complexaccording to claim 41, wherein the amine substituent of the R₅ alkylgroup has the structure:

wherein n is an integer from 7-23.
 43. The transfection complexaccording to claim Error! Reference source not found., wherein R₄ is astraight-chain alkyl group having 8 to 24 carbon atoms, R₅ is a propylgroup substituted with one amine and one alcohol, and R₇ is H.
 44. Thetransfection complex according to claim 43, wherein the aminesubstituent of the R₅ alkyl group comprises an aminoethanol, anaminopropanol or an aminobutanol group.
 45. The transfection complexaccording to claim 44, wherein the amine substituent of the R₅ alkylgroup has the structure:

wherein n is an integer from 7-23.
 46. The transfection complexaccording to claim Error! Reference source not found., wherein theliposome further comprises one or more neutral lipids.
 47. Thetransfection complex according to claim 46, wherein the neutral lipid isDOPE, DOPC or cholesterol.